Detection of Phytophthora ramorum in US commercial nurseries has led
to a number of quarantine regulations. Methods such as real-time PCR (RT-PCR)
provide rapid and reliable detection that can supplement attempts to culture
P. ramorum from symptomatic tissue. We adapted and optimized a previously
described mitochondrial gene-based RT-PCR assay for use with a Cepheid
SmartCycler v.1 and ready-to-use lyophilized PCR beads. The detection limit was
10 fg of P. ramorum genomic DNA. No cross-reactivity was observed on the
SmartCycler for seven additional Phytophthora species tested, which
included species known to cross-react in other assays as well as recently
described species Phytophthora foliorum and P. kernoviae. The
SmartCycler assay described here was used to detect P. ramorum in a
set of 2008 California field samples with a high degree of accuracy.

Introduction

When Phytophthora ramorum (Werres, De Cock & Man in’t Veld) sp.
nov., cause of significant oak mortality in the coastal forests of
California (15,16), was recovered from a California nursery in 2001, it
increased concerns that the pathogen could spread to uninfested regions of the
USA through movement of infected nursery stock. Despite regulatory efforts in
place in California, potentially infected material from a nationwide nursery
supplier was shipped to 783 garden centers in 39 states in 2004 (19). Within a
few weeks of the shipment, the United States Animal Plant Health Inspection
Service (APHIS) increased restrictions (1) and sampling for the national survey
to determine the pathogen’s distribution and to prevent its future spread.

Both nested PCR and real-time PCR (RT-PCR) have been developed for the
detection of P. ramorum (3,6,10,11,12,13,14,18,20,22,23,24). The internal
transcribed spacer (ITS) region of the nuclear ribosomal RNA gene has been
widely used (8,10,11,12,24), but cross-reactivity has been observed with P.
lateralis and/or P. hibernalis in these assays (3,6,10,12,20). The
gene sequences of b-tubulin and elicitin can be used for detection (2), but
P. lateralis has been shown to cross-react with the elicitin primers (2).
The spacer region between coxI and coxII of the
mitochondrial genome offers another detection target (13,22,23); this sequence
is abundant and variable at the species level (13,22). Using a RT-PCR assay
based on this region, no cross-reactivity was observed when tested with 45 other
Phytophthora species (22) or when a number of PCR assays for detection of
P. ramorum were compared in a multi-laboratory study using DNA from a
standardized set of isolates of different Phytophthora species (14).

The assay described by Tooley et al. (22) was optimized for the ABI Prism
7700 Sequence Detection System, SDS (Applied Biosystems, Foster City, CA). Here,
we report its adaptation to the Cepheid SmartCycler (Cepheid, Sunnyvale, CA).
The SmartCycler is widely used by molecular diagnostic laboratories certified as
part of the National Plant Diagnostic Network (www.npdn.org), so successful
adaptation of an assay to this platform will contribute to its nationwide
applicability. The goals of this study were to optimize assay conditions for the SmartCycler using ready-to-use lyophilized PCR beads, evaluate specificity by
testing DNA from Phytophthora species known to cross-react in other PCR
assays, and to test recently-described species Phytophthora foliorum (7)
and Phytophthora kernoviae (4) to confirm that no cross-reactivity
occurs in the assay.

Real-time PCR Conditions

Specific primers and probes used here were previously designed by Martin et
al. (13) and Tooley et al. (22), respectively (Table 1). RT-PCR reactions were
performed using the Cepheid SmartCycler v.1 and software 2.0 D (Cepheid,
Sunnyvale, CA). Background fluorescence was automatically determined by the
analysis software using raw data from cycles 3 to 10. Thresholds were set 10
standard deviations above the background. DNA samples were tested in a total
volume of 25 µl containing reconstituted 1X OmniMix HS (Cepheid, Sunnyvale, CA),
1 µM each P. ramorum primer, and 0.15 µM PrFAM probe. Duplex PCR
reactions also contained 0.1 µM each plant primer, 0.2 µM Plant CAL Red probe,
and an additional 1 µM MgCl2 and 75 µM each dNTP. Both plant and pathogen
probes were diluted and placed at -20°C prior to use. Cycling conditions for all
PCR reactions were 95°C for 2 min and 60 cycles of 95°C for 1 s and 60°C for 30 s. Fluorescence was recorded during each annealing/extension step. Detection of
CAL Red 610 was recorded through the ROX channel on the SmartCycler without
additional calibration.

Table 1. Polymerase chain reaction primera and fluorescent probe sequencesb
used in the RT-PCR assay for the detection of Phytophthora ramorum on the
Cepheid SmartCycler.

z Probes were labeled at the 5’ end with either the fluorescent reporter dye
6-carboxyfluorescine-aminohexyl amidite (FAM) or CAL Fluor Red 610 amidite (CAL
Red 610) and labeled at the 3’ end with a black hole quencher dye (BHQ,
Biosearch Technologies, Novato, CA).

Specificity of P. ramorum Primers and Probe

Genomic DNA samples from 21 isolates representing eight Phytophthora
spp. (Table 2) were tested with RT-PCR. DNA was extracted from lyophilized
tissue of P. ramorum, P. cactorum, and P. citricola
isolates grown on a liquid synthetic medium (25). A DNeasy Plant Mini kit
(Qiagen, Inc.,Valencia, CA) was used to extract DNA from the six P. ramorum
isolates. DNA from isolates 384, 385, and 422 was extracted following Goodwin et
al. (9). DNA from the remaining species was part of a multi-laboratory study
designed to compare different P. ramorum detection assays (14). DNA
concentrations for the samples extracted in our laboratory were determined using
the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Duplicate RT-PCR reactions for each isolate were tested twice using 100 pg of
template DNA along with primers FMPr1a and FMPr7 and the PrFAM probe. Sterile
distilled water was used as a negative control.

Table 2. Isolates of Phytophthora spp. used in this study with cycle
threshold (Ct) values from RT-PCR analysis using the Cepheid SmartCycler.

Species

Isolate no. (genotype)

Origin

Host

Mean
Ct value ± SEx

P. cactorum

384

New York
(W.
Wilcox)

Fragaria
x ananassa

>60 ± 0y

P. cactorum

385

New York
(W.
Wilcox)

Malus
sylvestris

>60 ± 0

P. cambivora

P0592z

Oregon

Abies procera

>60 ± 0

P. cambivora

P1432

Japan

Malus pumila

>60 ± 0

P. citricola

422

UK (D. Mitchell)

Cornus
sp.

>60 ± 0

P. citricola

P1817

South Africa

Madia sativa

>60 ± 0

P. foliorum

P10970

California

Rhododendron
sp.

>60 ± 0

P. foliorum

P10971

California

Rhododendron
sp.

>60 ± 0

P. hibernalis

P3822

Australia

Citrus sinensis

>60 ± 0

P. hibernalis

P6871

Portugal

Citrus
sp.

>60 ± 0

P. kernoviae

P10956

UK

Rhododendron
ponticum

>60 ± 0

P. kernoviae

P10957

UK

Rhododendron
ponticum

>60 ± 0

P. lateralis

P3888

Oregon

Chamaecyparis
lawsoniana

>60 ± 0

P. lateralis

P10177

Oregon

Chamaecyparis
lawsoniana

>60 ± 0

P. lateralis

P1728

Oregon

Chamaecyparis
lawsoniana

>60 ± 0

P. ramorum

CBS 101553 (EU1)

Germany (S. Werres, BBA 9/95)

Rhododendron
catawbiense

23.53 ± 0.12

P. ramorum

CBS 101331 (EU1)

Netherlands
(S.
Werres)

Rhododendron
sp.

24.15 ± 0.08

P. ramorum

CBS 101330 (EU1)

Netherlands
(S.
Werres)

Viburnum
sp.

25.52 ± 0.41

P. ramorum

Pr-52 (NA1)

California
(D.
Rizzo)

Rhododendron
sp.

23.65 ± 0.21

P. ramorum

5-C (NA1)

California
(N.
Shishkoff)

Camellia
sasanqua

25.12 ± 0.19

P. ramorum

288 (NA1)

California
(M.
Garbelotto)

Rhododendronsp.

25.15 ± 0.12

x For all isolates except the six P. ramorum isolates, Ct values
presented represent means of four observations, plus or minus standard error
(SE). Two experiments with two replications each were conducted. Ct values for
the six P. ramorum isolates represent means of eight observations; two
experiments with two replications each were conducted using DNA from two
separate extractions.

y No fluorescence was detected after 60 cycles of PCR amplification when
tested with 100 pg of DNA.

z Isolates with numbers beginning with the letter P originated from the World
Phytophthora Collection at the University of California-Riverside.

High specificity was observed using primers FMPr1a and FMPr7 and the PrFAM
probe in a RT-PCR assay with an annealing temperature of 60°C on the
SmartCycler. When multiple isolates of Phytophthora spp. were tested,
only the P. ramorum isolates, three from Europe and three from the United
States,
had cycle threshold (Ct) values ranging from 22.91 to 27.09; all other species
of Phytophthora and the water controls were not detected after 60 cycles
(Table 2).

Assay Sensitivity Using P. ramorum Genomic DNA

Three experiments were conducted with the SmartCycler using serial dilutions
of genomic DNA to determine assay sensitivity with and without the simultaneous
amplification of plant DNA. DNA was extracted from P. ramorum isolate 288
following Goodwin et al. (9) and quantified using the NanoDrop ND-1000
Spectrophotometer. Fresh serial dilutions (1 ng to 10 ag) were prepared with
sterile distilled water for each experiment. Dilution experiments were completed
three times, with two repetitions tested for each DNA sample or control.
Approximately 7 ng of genomic healthy azalea DNA and 100 pg of genomic P.
ramorum DNA of isolate 288 were tested as controls in the second and third
experiments. Sterile distilled water was used as a negative control in all
experiments. Standard curves were generated for each experiment using Sigma Plot
version 10.0 (Systat Software, Inc., Point Richmond, CA).

The first assay determined the limit of detection by use of a serial dilution
of P. ramorum DNA tested with P. ramorum-specific primers and
probe. Detection limits of the RT-PCR assay using P. ramorum primers and
probe were consistent and reproducible for DNA amounts between 1 ng and 10 fg.
Mean Ct values for these DNA concentrations were 18.02 to 35.40, respectively,
with values differing by approximately 3.49 cycles for each dilution. Although
detection for samples with less than 10 fg of DNA was possible (Fig. 1A), it was
variable and therefore omitted from the standard curve analysis (Fig. 1B). Mean
Ct values along with standard errors for 1 fg and 100 ag were 43.03 ± 3.43 and
46.36 ± 4.31, respectively. Amplification of water controls was not observed.

Fig. 1. Sensitivity of the mitochondrial gene region-based Phytophthora ramorum detection assay using the Cepheid SmartCycler. (A) Real-time PCR amplification profile for a representative dilution series of DNA extracted from Phytophthora ramorum isolate 288. (B) Standard curve of cycle threshold (Ct) values calculated from serial dilutions of P. ramorum DNA tested with the P. ramorum primer and probe set. (C) Standard curve of Ct values calculated from serial dilutions of P. ramorum DNA spiked with DNA extracted from a healthy azalea and tested with the P. ramorum primer and probe set. (D) Standard curve of Ct values calculated from serial dilutions of P. ramorum DNA spiked with DNA extracted from a healthy azalea and tested with both the P. ramorum and plant primer and probe sets. Three separate DNA dilutions were produced for each experiment and duplicate PCR reactions were tested with each dilution (n = 6). Sigma Plot version 10.0 (Systat Software Inc., Point Richmond, CA) was used for statistical analyses; standard error bars are indicated.

The second experiment determined the effect of adding plant DNA to a set of
similar PCR reactions. In this experiment, each PCR reaction containing a known
amount of diluted P. ramorum DNA received an addition of approximately 7
ng of DNA from a healthy azalea, Rhododendron 'Gloria.' Plant DNA was
extracted using the FastDNA Kit (Qbiogene, Inc., Carlsbad, CA) and quantified
using the NanoDrop ND-1000 Spectrophotometer. RT-PCR was performed with the
P. ramorum specific primers and probe. Sensitivity was minimally affected as
Ct values were similar to those values obtained from reactions without added
plant DNA (Fig. 1C). Additionally, the slopes of the standard curves for both
experiments were similar (-3.49 vs. -3.57). Neither water controls nor healthy
plant DNA samples produced amplification curves.

The third assay detected P. ramorum DNA in a duplex PCR reaction
containing both P. ramorum and plant primers and probes. In this
experiment, samples containing both diluted P. ramorum DNA and
approximately 7 ng of healthy 'Gloria' azalea DNA were tested. Sensitivity of
the duplex assay was determined using the pathogen and plant primer and probe
sets simultaneously. Minimal differences were observed between the FAM Ct values
from this test and the previous experiments; standard curves from all three
experiments were similar (Fig. 1D). All samples containing plant DNA amplified
with the plant primer and probe set. The mean CAL Red 610 Ct value and
corresponding standard error for samples containing between 10 ag and 100 pg of
P. ramorum DNA was 28.53 ± 0.16. For reactions containing 1 ng of P.
ramorum DNA, the mean Ct value with the plant probe was 34.72 ± 1.53 (SE).
Sterile water controls were negative for both pathogen and plant DNA. The P.
ramorum genomic DNA control amplified with only the pathogen primer and
probe set. Conversely, the healthy plant DNA only amplified with the plant
primer and probe set.

Dilution Series Experiments Using Inoculated Plant Material

Two separate DNA samples were extracted from infected leaves (two 6-mm
diameter leaf disks) of Rhododendron ‘Cunningham’s White’ using a
Qbiogene FastDNA Kit according to manufacturer’s instructions. Leaves had been
inoculated with P. ramorum isolate Pr-52 using the method described by
Tooley et al. (21). DNA samples were diluted with sterile water and tested using
the ABI Prism 7700 SDS (22). Dilutions were stored at -20°C with limited
freeze-thaw cycles and tested using the SmartCycler. Each dilution was tested in
duplicate RT-PCR reactions using specific P. ramorum primers and probe.
Controls of sterile water, healthy azalea DNA (approximately 7 ng), and genomic
P. ramorum DNA (100 pg) from isolate CBS 101553 were also tested.
Detection was possible with all dilutions and with the positive genomic DNA
control, and no amplification of the water control was observed. At the lowest
dilution of 10-6, the pathogen was detected with a mean Ct value of 49.79 ± 5.92
(SE) (Table 3). Using the equation of the standard dilution series curve (Fig.
1B) the initial sample was predicted to contain ca. 201 pg of P. ramorum
DNA.

x DNA was extracted from two 6-mm diameter leaf disks using a
Qbiogene
FastDNA Kit according to manufacturer’s instructions.

y Ct values are means of four observations, plus or minus standard
error
(SE). Two separate extractions were performed (each using
two 6-mm diameter leaf
disks), and two replicate RT-PCR
experiments were conducted (n = 4).

z ND = not determined due to out of range of the standard curve.

Detection Using Field Samples from California

In a blind study, DNA from 16 field samples obtained from a variety of hosts
in California in 2008 was tested using our duplex RT-PCR assay (Table 4).
Samples were provided by the California Department of Food and Agriculture
(CDFA), extracted using the Qiagen DNA Easy kit, and evaluated prior to our
testing using culture techniques, nested PCR (8,10), and/or RT-PCR (12,24). In
our laboratory, these DNA samples were diluted ten-fold and tested in duplicate
RT-PCR reactions using 2 µL of freshly diluted template DNA. At least two
dilutions were tested for each sample (Table 4). Controls for each RT-PCR
experiment included DNA from a healthy plant, a P. ramorum-infected
plant, and a P. ramorum isolate.

Table 4. Duplex RT-PCR results for plant samples collected from the field in
California in 2008 and tested in a blind study.

No.

Host

Detection with
plant primers

Detection with P.
ramorum primers

CDFA deter-
minationu

Mean Ct value±SEv

Resultw

Mean Ct value±SE

Result

Result

Method

1

Arctostaphylos otayensis

23.51 ± 0.28

+

60.00 ± 0.00

−

+

RT-PCR

2

Camellia 'Debutante'

23.80 ± 0.19

+

22.29 ± 0.01

+

+

culture

3

Camellia sasanqua

23.39 ± 0.17

+

32.90 ± 0.33

+

+

culture

4

Heteromeles arbutifolia

23.24 ± 0.11

+

60.00 ± 0.00

−

−

RT-PCR

5

Kalmia latifolia

24.52 ± 0.05

+

60.00 ± 0.00

−

−

RT-PCR

6

Laurus nobilis

21.66 ± 0.15

+

60.00 ± 0.00

−

−

RT-PCR

7

Magnolia grandiflora

22.94 ± 0.18

+

60.00 ± 0.00

−

−

RT-PCR

8

Photinia fraseri

21.75 ± 0.12

+

60.00 ± 0.00

−

−

RT-PCR

9

Rhamnus californica

22.26 ± 0.10

+

60.00 ± 0.00

−

−

RT-PCR

10

Rhododendron 'Doctor Arnold Endtz'

26.43 ± 0.19

+

39.00 ± 0.31

+

+x

nPCR and culture

11

Rhododendron 'Nova Zembla'

23.02 ± 0.65y

+

51.55 ± 2.75

suspect

+

nPCR

12

Rhododendron 'PJM'

24.57 ± 0.12

+

60.00 ± 0.00

−

−

RT-PCR

13

Umbellularia californica

23.73 ± 0.20

+

34.61 ± 0.11

+

+

RT-PCR

14

Umbellularia californica

22.64 ± 0.14

+

34.62 ± 0.22

+

+

RT-PCR

15

Umbellularia californica

23.10 ± 0.14

+

29.13 ± 0.11

+

+

RT-PCR

16

Umbellularia californica

24.01 ± 0.08z

+

57.80 ± 2.20

−

−

RT-PCR

u Samples were collected from March to December 2008 in California and were
previously processed by the California Department of Food and Agriculture using
the Qiagen DNA Easy kit. The following methods were used to test for P.
ramorum: real-time PCR (RT-PCR, APHIS-approved protocol) (12,24), culture,
and nested PCR (nPCR) (8,10).

v Except where noted, Ct values are means of four observations, plus or minus
standard error (SE). Two experiments with two replications each were conducted.

w Detection of the target sequence is positive (+) if the threshold was
consistently crossed in each duplicated RT-PCR reaction. Negative (−) results
indicate that fluorescence was not detected from multiple reactions after 60
cycles. A sample was considered suspect if it produced fluorescence after 45
cycles in duplicated reactions.

x Positive results were obtained using both culture and nested PCR.

y n = 6.

z n = 7.

All 16 field samples amplified with the plant primers and probe producing
mean Ct values ranging from 21.66 to 26.43 (Table 4). Six of the field samples
consistently amplified with the P. ramorum primers and probe in each
reaction. Mean Ct values for these samples ranged from 22.29 to 39.00. No
fluorescence was detected after 60 cycles for eight of the field samples. For
sample no. 11, the results agreed within replications but not between
repetitions. In a third repetition both reactions were positive, however.
Amplification was also observed in one of the reactions of sample no. 16, but
not in the other reactions. Another repetition was run with triplicate RT-PCR
reactions; no amplification was observed. Results for fourteen out of 16 samples
agreed with those from the CDFA (Table 4).

Conclusions

The previous assay conducted on the ABI instrument amplified only P.
ramorum and not 45 other species of Phytophthora tested (22). Despite
modifications made to this assay to adapt it to the Smartcycler, P. cactorum,
P. cambivora, P. foliorum, P. hibernalis, and P. lateralis
did not amplify in our assay even though they have shown some degree of
cross-reactivity in other PCR assays for detection of P. ramorum
(2,3,5,6,7,11,12,20). Cross-reactivity was also not observed with the recently
described P. kernoviae (4). We were unable to test all species of
Phytophthora for cross-amplification in this assay, but believe because
these most closely-related species of Phytophthora did not amplify, that
other known species are also unlikely to cross-react. Actual verification of
this hypothesis would require testing of all known Phytophthora species.

Similar to the results of Schaad et al. (17), the sensitivity of this assay
was slightly altered when it was adapted from the ABI Prism 7700 SDS to the
SmartCycler. Using serial dilutions of P. ramorum genomic DNA, the
consistent detection limit on the ABI Prism 7700 SDS was determined to be 1 fg
(22), while the limit found on the SmartCycler was 10 fg. Although DNA amounts
less than 10 fg of P. ramorum DNA were detected by the SmartCycler,
results were inconsistent and variable. The actual limit of detection is
probably less than 10 fg, but values between 1 and 10 fg were not tested.

A limitation of our dilution series experiments was that we performed them
from a single extraction of P. ramorum isolate 288 and did not truly
replicate the entire detection assay starting with isolate culturing and DNA
extraction. Thus, due to possible differences in mitochondrial numbers that may
exist between different growth replications, some experimental variation that
may have existed was not accounted for in our experiments. However, this
potential variation, if present, unlikely had a significant impact on our
assay's sensitivity. When two sets of DNA dilutions were extracted from
inoculated rhododendron leaf disks and tested with RT-PCR, little variation was
observed. The lowest amount detected by the SmartCycler was ca. 2 fg of P.
ramorum DNA [(22) and this study], a value similar to the one determined by the ABI Prism 7700 SDS.

While assay sensitivity decreased when it was transferred to the SmartCycler,
assay efficiency increased. Using the equation E = 10(-1/S) - 1, where S is the
slope, PCR amplification efficiency (E) was determined (12). By transferring the
assay to the SmartCycler, the efficiency of the RT-PCR reactions increased from
approximately 87% [based on the slope of -3.68, (22)] to 93% (based on the slope
of -3.49). However, adding plant DNA to each reaction reduced the efficiency to
approximately 90%; approximately 84% in a duplex reaction. While pathogen Ct
values were slightly affected by dual amplification, detection limits remained
constant and are comparable to those previously observed with the ABI Prism 7700
SDS (22).

In a blind study, we tested field samples from California in 2008 previously
evaluated at CDFA and found agreement in 14 of 16 samples. Possible explanations
for lack of agreement for certain samples include variation in the amount of
target DNA present, the potential presence of PCR inhibitors, potential
degradation of the DNA sample, and/or the specificity and sensitivity of RT-PCR
assays. The results demonstrate the importance of using multiple detection
methods to obtain the most accurate diagnoses.

The previously described (22) RT-PCR assay based on a mitochondrial gene
region has been successfully transferred to the SmartCycler platform, and is
ready for extensive laboratory and field applications. This modified assay will
provide an additional specific and sensitive tool for monitoring plant material
for the presence of P. ramorum to limit its potential spread to new
areas.

Acknowledgment

We are very grateful to Cheryl Blomquist at the California Department of Food
and Agriculture for providing the field samples used in this study.

24. USDA, APHIS PPQ CPHST. 2005. Quantitative multiplex real-time PCR
(qPCR) for detection of Phytophthora ramorum using a TaqMan
system on the Cepheid SmartCycler and the ABI 7900/7000. Document
Control Number WI-B-T-1-6.10-26-2005.